The epidermis of humans and animals is composed of three layers: the stratum basale, the stratum spinosum and the stratum corneum, as best shown in FIG. 1. The stratum basale, the innermost layer, is the only layer in which active cell division occurs. When division transpires, a daughter cell, called a keratinocyte, begins to undergo terminal differentiation. By this process, the keratinocyte is sequentially transformed into a cell of the spinosum and then into one of the corneum. Among the changes occurring during differentiation are increased synthesis of ceramide, loss of the nucleus, cell death and replacement of the cytoplasm with keratin. As keratinocytes reach the outermost stratum corneum layer, they are shed to the environment.
The stratum corneum provides a mechanical barrier to the external environment, and the concentration of ceramide in this layer is directly correlated with moisture retention. Consequently, agents which increase ceramide content will also improve moisture retention. Further, acceleration of keratinocyte differentiation increases the turnover rate of the stratum corneum, thereby minimizing the appearance of excessively dry skin, known as xerosis.
The biochemical sequence of events in keratinocyte differentiation is complex and poorly understood. Regardless of the details, it appears that agents which induce the synthesis of ceramide (a sphingolipid), as shown in FIG. 2, are associated with the induction of keratinocyte differentiation. Such agents include UVA radiation (Maziere, et al. Biochemical and Biophysical Research Communications, 281(2):289-294, 2001), L-lactic acid (Rawlings, et al. Archives of Dermatological Research, 288(7):383-390, 1996), lactic acid bacteria (DiMarzio, et al. Journal of Investigative Dermatology, 113(1): 98-106, 1999) and extracellular calcium (Watanabe, et al, J. Biol. Chem., 273 (16): 9651-5, 1998).
L-lactic acid, but not D-lactic acid, has recently been used as an effective treatment for xerosis. It is most likely efficacious because it promotes keratinocyte differentiation and ceramide synthesis (Rawling, et al; Arch. of Derm. Res., 288(7):383-90, 1996). The stereospecificity suggests that the action of L-lactic acid is not simply one of pH adjustment. Rather, the observation that the carbon label from L-lactic acid is incorporated into ceramide suggests that L-lactic acid provides an increased substrate for ceramide synthesis.
The normal pH of the stratum corneum is somewhat acidic, 5.80 in men and 5.54 in women (Ehlers, et al. Skin Research and Technology, 7(2):90-4, 2001). Maintenance of normal skin flora is pH dependent, and even modest increases in pH (0.1 to 0.2 pH units) are sufficient to foster growth of undesirable microorganisms such as Candida albicans (Runeman, et al. Acta Dermato-Venereologica 80(6): 421-4, 2000). Therefore, it is important that products designed for application to the skin have carefully controlled pH levels.
It has been found that the membranes of human keratinocytes possess a calcium-sensing protein that responds to increases in extracellular calcium, thereby initiating terminal differentiation (Oda, et al. Journal of Biological Chemistry, 275(2): 1183-90, 2000). Furthermore, researchers have shown that the addition of calcium to culture media will initiate in vitro differentiation in cultured human keratinocytes by activating a number of protein kinases, and calcium is thought to act very early in the differentiation process (Schmidt, et al. Journal of Biological Chemistry, 275(52):41011-7, 2000).
Current methods for the stimulation of more youthful skin cells and for promoting the appearance of younger skin include skin peels, such as those utilizing glycolic acid. The pH of such peels may be as low as 2.0, a hydrogen ion concentration more than 3000 times greater than that measured on the surface of normal skin. Thus, it is not surprising that the use of glycolic acid peels may be accompanied by redness and irritation. Alternatively, retinoid compounds have been used topically for their ability to stimulate keratinocyte differentiation and promote collagen and elastin synthesis. However, they may also induce photosensitivity, limiting their utility in susceptible individuals.
For these reasons, there remains a need in the art for a method for stimulating more youthful looking skin without producing undesirable side effects, and which would also be useful for accelerating the healing of wounds without encouraging the growth of undesirable organisms such as Candida albicans. 